Bifidobacterium longum ncimb 41676

ABSTRACT

Bifidobacterium longum  strain NCIMB  41676  improves sleep.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 16/794,019, filed onFeb. 18, 2020, issued as U.S. Pat. No. 10,813,366, which is acontinuation of International Application No. PCT/EP2019/052140, filedon Jan. 29, 2019, each incorporated by reference herein in its entirety,PCT/EP2019/052140 claiming the benefit of priority to EuropeanApplication No. 18153997.4, filed on Jan. 29, 2018.

INTRODUCTION

Accumulating data indicate that the gut microbiota communicates with thecentral nervous system (CNS), the part of the nervous system consistingof the brain and spinal cord,—possibly through neural, endocrine andimmune pathways—and thereby influences brain function and behaviour(Dinan et al., 2013; Bravo et. al, 2011). Probiotics are defined as livebacteria, which when ingested in adequate amounts, confer a healthbenefit (WHO, 2001). Recent evidence suggests that probiotics may have arole in mental health and well-being (Dinan et al., 2013). However,while there is increasing interest in this field and preclinical trialsshow promising results, this field is in its infancy and very little isknown about the effect of probiotic on mental well-being in humans.

Sleep is vital for normal physiological and psychological functioning.Unfortunately, 50-70 million Americans experience disordered sleep,inflicting substantial financial and societal cost (Institute ofMedicine Committee on Sleep & Research, 2006). Sleep disturbance is asignificant stressor that contributes to cognitive impairment, affectivedisruption and physical disease Modern lifestyles contribute to thisgrowing health concern by shifting importance away from healthy sleephabits and towards other activities. Developing effective and safetreatments for disturbed sleep is a necessary public priority.

Pharmaceutical sleep aids, like most pharmaceutical treatments, can haveharmful side effects. Psychological treatments are generally side-effectfree but require substantial time and effort.

Probiotics may have considerable clinical utility in helping addressdisrupted sleep. However, the relationship between probiotics and sleepis still poorly understood and no studies have reported assessing theeffects on sleep in humans of a Bifidobacterium longum strain.

WO2010/0606722 A1 reported, based on testing with mice, that aBifidobacterium longum BAA-999 may improve sleep quality and reduce thenumber of episodes of wakefulness in infants, and hypothesised that thiswas due to possible anxiolytic effects in humans. However, as reportedin Pinto-Sanchez et al., 2017(3) further work in humans did not supportthis anxiolytic effect.

There is a need for a probiotic which is effective in improving sleepquality.

STATEMENTS OF INVENTION

The invention also provides Bifidobacterium longum strain NCIMB 41676for use in improving sleep in humans.

According to the invention there is provided Bifidobacterium longumstrain NCIMB 41676 for use in improving sleep quality in humans.

According to the invention there is provided Bifidobacterium longumstrain NCIMB 41676 for use in improving sleep quality in mammals.

According to the invention there is provided Bifidobacterium longumstrain NCIMB 41676 for use in improving sleep in mammals.

The Bifidobacterium strain NCIMB 41676 may be in the form of viablecells.

The Bifidobacterium strain NCIMB 41676 may be in the form of non-viablecells.

The Bifidobacterium strain NCIMB 41676 may be present in the formulationin an amount of more than 10⁶ cfu, typically from 10⁷ to 10¹⁰, typicallyfrom 10⁸ to 10⁹ cfu. In one case the Bifidobacterium strain NCIMB 41676is present in the formulation in an amount of about 1×10⁹ cfu.

Bacterial viability reflects the number of culturable bacteria within asample, i.e. the number of bacteria which retain the ability toreproduce when grown under optimal conditions (Viable cells). Putanother way viability reflects the number of individual bacterial cellswhich retain the ability to replicate into larger bacterial colonies(colony forming units (CFUs)).

Viability is commonly determined using plate-counting methods, whereby abacterial sample is diluted and then incubated on an agar platecontaining the necessary nutrients for growth. Viability is thencalculated from the number of bacterial colonies identified on a plate.Such methods are summarized in Modern Food Biology 2005 7^(th) edition,James Monroe Jay, Martin J. Loessner, David A. Golden, Springer Science,New York.

Whilst plate-counting gives a good indication of viability, it does notencompass all living bacterial cells in the sample. (Kell, Douglas B.,et al. “Viability and activity in readily culturable bacteria: a reviewand discussion of the practical issues.” Antonie van Leeuwenhoek 73.2(1998): 169-187).

Samples will also contain “viable but non-culturable” (VBNC) cells whichremain metabolically active but have lost the ability to replicate atthe time of analysis by plate count, and thus despite being alive willnot form CFUs. Finally, samples will also contain dead cells. These twogroups can be grouped together as “Non-Viable cells”. ThereforeNon-viable cells are the inverse of Viable cells i.e. all those cellswhich have lost the ability to replicate when tested.

All samples containing Viable cells will also contain Non-Viable cells,therefore the definition of a Viable cell culture is clarified using CFUmeasurements.

All Non-Viable samples will contain at least VNBCs and possibly smallamounts of Viable cells. Industry standard lower level detection limitsof 10³ CFU/g viable cells allow for the inherent process variabilitycaused by the presence of a certain number of VBNCs/Viable cells inNon-Viable samples.

In some embodiments, such as, but not limited to, special sterile foodproducts or medicaments a non-replicating form of a probiotic strain maybe preferable. For example, at least 95%, preferably at least 97%, morepreferably at least 99% of the Bifidobacteria strain can benon-replicating in the composition.

In some embodiments the formulation is in the form of a bacterial broth.

In some cases the formulation in the form of a freeze dried powder.

The formulation may further comprise a prebiotic material.

In some cases the formulation further comprises an ingestible carrier.The ingestible carrier may be a pharmaceutically acceptable carrier suchas a capsule, tablet or powder. Alternatively, the ingestible carrier isa food product such as acidified milk, a yoghurt, a frozen food such asfrozen yoghurt or ice cream, a gum, a candy, a milk powder, a milkconcentrate, a cheese spread, a nutritional composition, a nutritionalsupplement, a cereal bar, a dressing or a beverage.

In one case the formulation is an infant food.

In some embodiments the formulation is in the form of a fermented foodproduct.

The formulation may be in the form of a fermented milk product.

In some cases the carrier does not occur in nature.

The formulation may further comprise a protein and/or peptide, inparticular proteins and/or peptides that are rich inglutamine/glutamate, a lipid, a carbohydrate, a vitamin, mineral and/ortrace element.

The formulation may further comprise an adjuvant.

The formulation may further comprise a bacterial component.

The formulation may further comprise a drug entity.

The formulation may further comprise a biological compound.

The strains are in the form of freeze dried powder which is blended withfood grade excipient, and filled into a format such as a sachet orcapsule.

The strain of the invention may be administered to animals (includinghumans) in an orally ingestible form in a conventional preparation suchas capsules, microcapsules, tablets, granules, powder, troches, pills,suppositories, suspensions and syrups. Suitable formulations may beprepared by methods commonly employed using conventional organic andinorganic additives.

The amount of active ingredient in the medical composition may be at alevel that will exercise the desired therapeutic effect.

The formulation may also include, a drug entity or a biologicalcompound.

In addition a vaccine comprising the strains of the invention may beprepared using any suitable known method and may include apharmaceutically acceptable carrier or adjuvant.

The introduction of probiotic organisms is accomplished by the ingestionof the micro-organism in a suitable carrier. It would be advantageous toprovide a medium that would promote the growth of these probioticstrains in the large bowel. The addition of one or moreoligosaccharides, polysaccharides, or other prebiotics enhances thegrowth of lactic acid bacteria in the gastrointestinal tract. Prebioticsrefers to any non-viable food component that is specifically fermentedin the colon by indigenous bacteria thought to be of positive value,e.g. bifidobacteria, lactobacilli. Types of prebiotics may include thosethat contain fructose, xylose, soya, galactose, glucose and mannose. Thecombined administration of a probiotic strain with one or more prebioticcompounds may enhance the growth of the administered probiotic in vivoresulting in a more pronounced health benefit and is termed synbiotic.

It will be appreciated that the probiotic strains may be administeredprophylactically or as a method of treatment either on their own or withother probiotic and/or prebiotic materials as described above. Inaddition, the bacteria may be used as part of a prophylactic ortreatment regime using other active materials such as those used fortreating related conditions especially those with an immunologicalinvolvement. Such combinations may be administered in a singleformulation or as separate formulations administered at the same ordifferent times and using the same or different routes ofadministration.

The strains of the invention may be formulated to facilitate controlledrelease such as a delayed release of the strain. For example, theformulation may be adapted to release the strain at a particularlocation in the gastrointestinal tract such as the small intestine or inthe colon. To achieve such a controlled release the strain may beformulated in a capsule which has a coating which is adapted to releasethe strain at a particular location. A range of coatings are availableto facilitate such controlled release. One such family of coatings arethose available under the Trademark Eudragit.

Sleep disturbances are observed at any stage of the life. Thesedisturbances are typically characterized by a decrease in the ability toinitiate and maintain sleep, and by a reduced proportion of the deeper,more restorative sleep. Quality of life is substantially impaired inindividuals suffering from those alterations.

Infant sleep normally changes over the first months of life to follow adiurnal rhythm with sleep lasting for a long unbroken period at nightand, similarly, sleep states change from being equally distributedbetween REM (active) and NREM (quiet) sleep at birth to one third REMand two thirds NREM by 8 months of age. Any failure to successfullynegotiate these changes in infancy can also have lasting effects on thesleep patterns of the child.

The most common sleep disturbances in infants and children are thoserelated to wakefulness (i.e. either difficulties in settling at bedtimeor failure to sleep through the night without interruptions). It hasbeen estimated that these disturbances affect 15 to 35% of infants agedless than 24 months. Infant and child sleep disturbances inevitably leadto parental sleep disturbance and stress which may result inadequatechild-parent interaction which in turn aggravates infant and childsymptoms leading to a vicious circle.

At the other end of life normal aging is also accompanied by changes inthe sleep quality, quantity, and architecture. Specifically, thereappears to be a measurable decrease in the ability of the healthyelderly to initiate and maintain sleep, accompanied by a decrease in theproportion of the deeper, more restorative NREM sleep.

Sleep is particularly important in older people. Along with the physicalchanges that occur as we get older, changes to our sleep patterns are apart of the normal aging process. As people age they tend to have aharder time falling asleep and more trouble staying asleep than whenthey were younger. It is a common misconception that sleep needs declinewith age. In fact, research demonstrates that our sleep needs remainconstant throughout adulthood. Changes in the patterns of our sleep,“sleep architecture”, occur as we age and this may contribute to sleepproblems. Sleep occurs in multiple stages including dreamless periods oflight and deep sleep, and occasional periods of active dreaming (REMsleep). The sleep cycle is repeated several times during the night andalthough total sleep time tends to remain constant, older people spendmore time in the lighter stages of sleep than in deep sleep.

Many older adults, though certainly not all, also report being lesssatisfied with sleep and more tired during the day. Studies on the sleephabits of older Americans show an increase in the time it takes to fallasleep (sleep latency), an overall decline in REM sleep, and an increasein sleep fragmentation (waking up during the night) with age. Theprevalence of sleep disorders also tends to increase with age. Researchsuggests that sleep disturbance among the elderly can in part beattributed to physical and psychiatric illnesses and the medicationsused to treat them.

In addition to changes in sleep architecture that occur as we age, otherfactors affecting sleep are the circadian rhythms that coordinate thetiming of our bodily functions, including sleep. For example, olderpeople tend to become sleepier in the early evening and wake earlier inthe morning compared to younger adults. This pattern is called advancedsleep phase syndrome. The sleep rhythm is shifted forward so that 7 or 8hours of sleep are still obtained but the individuals will wake upextremely early because they have gone to sleep quite early. The reasonfor these changes in sleep and circadian rhythms as we age is notclearly understood.

Environmental stressors can also be an issue. Exposure to stressnegatively affects sleep and the sleep/wake cycle. For example,experiencing work-related stressors having low social support, orexposure to trauma/combat can all disrupt sleep and the sleep/wakecycle.

Clinical trials in sleep medicine cover a wide range of sleep-wakeproblems, and accordingly the selection of outcome measures in sleepmedicine clinical trials needs to be tailored to the specific disorderunder examination. The investigator needs to consider the relativemerits choosing a self-report questionnaire versus a physiologic test.Surprisingly, the self-report measures are often more sensitive totreatment effects as compared with more expensive physiologic tests.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more clearly understood from the followingdescription thereof, given by way of example only, in which:

FIG. 1 is a graph illustrating the effect of B. longum 1714™ strain onsleep in an on-line study with healthy volunteers.

FIG. 2 is a study flow diagram of double-blinded, randomised,placebo-controlled, cross-over study of Bifidobacterium longum 1714 in achronic exam stress model in healthy individuals;

FIG. 3 is a graph of the global score of sleep quality measured byPittsburgh Sleep Quality Index (PSQI) at baseline and during exam stresscomparing the effect of probiotic and placebo administration;

FIGS. 4(a) and 4(b) are graphs of the sub-scores of the componentswithin PSQI that measure subjective sleep quality (FIG. 4(a)) and sleeplatency (FIG. 4(b)) at baseline and during exam stress comparing theeffect of probiotic and placebo administration;

FIGS. 5(a) and 5(b) are graphs of the sub-score of the component withinPSQI that measures sleep duration at baseline and during exam stresscomparing the effect of probiotic and placebo administration (FIG. 5(a))and change in sleep duration before and after intervention (FIG. 5(b));

FIG. 6 is a schematic diagram of an open label combo IBS studyschematic;

FIGS. 7(a) and 7(b) are plots of global PSQI score for the IBS patients(FIG. 7(a)) and IBS patients (PSQI over 5) (FIG. 7(b)) before and aftercombo treatment;

FIGS. 8(a) and 8(b) are plots of subjective sleep latency for the IBSpatients (FIG. 8(a)) and IBS patients (PSQI over 5) (FIG. 8(b)) beforeand after combo treatment;

FIGS. 9(a) and 9(b) are a plot and table, respectively, of subjectivesleep quality for the IBS patients before and after combo treatment; and

FIGS. 10(a) and 10(b) are a plot and table, respectively, of subjectivesleep quality for the IBS patients (PSQI over 5) before and after combotreatment.

DETAILED DESCRIPTION

A deposit of B. longum 1714™ strain was made at the National Collectionsof Industrial and Marine Bacteria Limited (NCIMB) Ferguson Building,Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, UK on Nov.5, 2009 and accorded the accession number NCIMB 41676.

WO2011/05853A, the entire contents of which are incorporated herein byreference, describes B. longum 1714™ strain—NCIMB 41676.

In the following examples NCIMB 41676 is tested in healthy humans,healthy humans involved in exams (a stressor that can lead to temporarydisrupted sleep patterns) and in IBS patients who suffer from thechronic effects of gastrointestinal disturbance with frequently reportedeffects on sleep.

EXAMPLE 1 Effect of B. Longum 1714™ on Sleep in Healthy Humans

42 subjects were recruited from the general population to take part inan on-line study. Subjects were provided in a capsule format containing1×10⁹ colony-forming units B. longum 1714™ for 5 weeks. Subjects wereasked to take 1 capsule per day for the duration of the study andcomplete an on-line questionnaire.

In the on-line study, participants were asked the following questionevery week:

Reflecting over the last week how would you rate your sleep quality?(0=very bad, 9=very good)

The results of this study showed that sleep quality increasedprogressively over the course of the study (FIG. 1). This work showsthat B. longum 1714™ strain increased sleep quality over the course ofthe study.

The invention is not limited to the embodiments hereinbefore describedwhich may be varied in detail.

EXAMPLE 2 A Double-Blinded, Randomised, Placebo-Controlled, Cross-OverStudy: Bifidobacterium Longum 1714™ Strain Improves Sleep During ChronicExam Stress in Healthy Individuals

We saw an improvement in sleep quality in the general healthy population(FIG. 1) therefore we assessed sleep quality after Bifidobacteriumlongum 1714 strain administration in healthy students in a chronicstress model in the controlled setting of a double-blinded, randomised,placebo-controlled, cross-over study. This human study utilising thenaturalistic stressor, exam stress, was performed to determine theusefulness of the Bifidobacterium longum 1714 as a means to improvesleep in a model of chronic stress (university exam stress of 3 weekperiod). The study outline is described in FIG. 2. Other studies haveshown that sleep quality is reduced by exams in students. The trialdesign used is a valid approach to assess sleep quality in chronicallystressed situations.

Study population included 20 healthy male and female student volunteersaged between 18-24 years. g*Power was utilised for power calculations,with an a-error probability (false positive) of 0.05, a power of 80% andeffect sizes of 0.3. The criteria for inclusion were: 1) able to givewritten informed consent; 2) age between 18 and 24 years; and 3) ingenerally good health as determined by the investigator. The exclusioncriteria were: 1) less than 18 and greater than 40 years of age; 2) havea significant acute or chronic coexisting illness, immunological,psychiatric, neurodevelopmental disorders, immunological, metabolicdisorders, or any condition which contraindicates, in the investigatorsjudgment, entry to the study; 3) have a condition or taking a medicationthat the investigator believes would interfere with the objectives ofthe study, pose a safety risk or confound the interpretation of thestudy results; all psychoactive medications, laxatives, enemas,antibiotics, anti-coagulants, over-the counter non-steroidalanti-inflammatories (NSAIDS) (subjects should have a wash-out period of4 weeks); 4) Current prebiotic or probiotic use; 5) Females who arepregnant or planning a pregnancy; 6) Are a current or past smoker; and7) Have a malignant disease or any concomitant end-stage organ disease.

The products administered during intervention were either probiotic B.longum 1714 (1×10⁹ CFU/day) in excipient or placebo consisting onlyexcipient in capsule format.

Pittsburgh Sleep Quality Index (PSQI) questionnaire was administered atbaseline and after the feeding phase to assess sleep quality over theprior month. The PSQI is a self-report measure comprised of 19 itemswhich are designed to measure seven key components indicatingproblematic or non-problematic sleep; sleep latency (time to fallasleep), sleep duration, sleep efficiency, sleep disturbances,subjective sleep quality, use of sleep medication, and daytimedysfunction due to sleep disturbance. Scores on each component arecombined to give a global score with >5 indicating significantdisturbance of sleep quality during the prior month.

The overall sleep quality reflected in the global score of PSQI atbaseline (before intervention) were similar between probiotic andplacebo groups. After 8 weeks of intervention, during exam stressperiod, the PSQI was measured again to compare the effect of probioticand placebo administration on sleep quality. Only placebo group hadworsened sleep quality (higher score) while there was no change in sleepquality in the probiotic group under exam stress (FIG. 3).

This result corresponds to those of the sub-scores of the componentswithin PSQI that measure subjective sleep quality and sleep latency(FIGS. 4(a) and (b)). Only placebo group had worsened subjective sleepquality and sleep latency (statistically significant higher scores)under exam stress compared to baseline.

Sleep duration as determined by the following question ‘How many hoursof actual sleep did you get at night?’ were similar in probiotic andplacebo groups at baseline. However, following 8 weeks of feeding withthe 1714 strain, there was an increase in subjective assessment of sleepduration in the probiotic group compared to placebo (FIGS. 5(a) and(b)). As graphed, a lower score reflects an improvement in sleepduration which is based on the correct analysis of the PSQIquestionnaire.

Taken together these results show that exam stress period affects sleepquality as shown in higher global PSQI score and higher sub-scores ofsubjective sleep quality, sleep latency and sleep duration suggestive ofloss of sleep efficiency in placebo group; and administration ofBifidobacterium longum 1714 for 8 weeks pre-exam stress improved sleepduration, efficiency and prevented the negative effect of the stressfulperiod on sleep quality.

EXAMPLE 3 An Open Label Irritable Bowel Syndrome (IBS) Study: ACombination B. Longum Product Shows Improvement in Sleep Quality asmeasured by the Pittsburgh Sleep Quality Index in IBS Patients

A human open label study was performed as follows to investigate theeffect of a combination of B. longum 1714™ and B. longum 35624® strainin adults with irritable bowel syndrome (IBS). The effects seen in thehealthy population (examples 1 and 2) with the 1714™ strain were furtherassessed in combination with the 35624® strains using the PittsburghSleep Quality Index (PSQI).

The clinical protocol for this trial was as follows:

Forty female subjects aged 18-55 years were recruited with IBS,diagnosed by Rome III criteria, and mild to moderate anxiety and/ordepression as determined by the Hospital Anxiety and Depression scale(HADs). Subjects who had a psychiatric diagnosis other than anxiety ordepression, a major inflammatory disorder or were on anti-depressants,anxiolytics or antipsychotics in the last 6 months were excluded. The‘COMBO’ (5×10⁸ CFU/day B. longum 35624+1×10⁹ CFU/day B. longum 1714),sachet format with maltodextrin and a flowability agent such as silicondioxide was taken for 8-weeks, followed by an 8 week washout (FIG. 6).The PSQI was measured at 0, 4, 8, 12 and 16 weeks. Sleep quality wasassessed through a modified PSQI designed to measure sleep quality anddisturbance over the past month in clinical populations as it hasacceptable reliability and validity (Buysse et al., 1989). The PSQI is a19-item self-report measure assessing sleep quality across sevendomains. Each of the sleep components yields a score ranging from 0 to3, with 3 indicating the greatest dysfunction. The sleep componentscores are summed to yield a total score ranging from 0 to 21 with thehigher total score (referred to as global score) indicating worse sleepquality. In distinguishing good and poor sleepers, a global PSQIscore >5 yields a sensitivity of 89.6% and a specificity of 86.5%.1(Buysse et al., 1991; Herman et al., 2002; Fillingim et al.; 2011, Portoet al., 2011). We used this >5 global PSQI cut off to look at asubpopulation of our IBS patients who have poor sleep quality.Subjective sleep quality is scored as follows ‘very good’ (0,) ‘fairlygood’ (1), ‘fairly bad’ (2) ‘very bad’ (3).

The daily ingestion of the combo product significantly decreased PSQIglobal scores at week 4 and week 8 (FIG. 7(a)). A decrease in PSQIglobal score indicates an improvement in sleep quality. When looking ata sub group of our IBS patients who had poor sleep quality (>5 globalPSQI) the combo significantly decreased PSQI global scores at all timepoints even those post feeding (FIG. 7(b)). The global PSQI score is thesum of seven component scores (sleep disturbance, overall sleep quality,sleep latency (time to fall asleep), duration of sleep, daytimedysfunction due to sleepiness, sleep efficiency, and need for medicinesto sleep). Among them, the combo significantly decreased the PSQI-sleeplatency score at week 4 and week 8, and 8 weeks post feeding (FIG.8(a)). Again, when we looked at a sub population of “bad sleepers” wesaw a decrease in PSQI-sleep latency at 8 weeks post feeding (FIG.8(b)). Another notable result was that when subjects were asked thequestion ‘during the past month, how would you rate your sleep qualityoverall?), the results showed that administration of the Combo productsignificantly improved sleep quality in agreement with the PSQI globalscore. Subjective sleep quality scores improved from baseline to the endof the intervention (8 weeks), in the combo group and maintained theireffect after feeding had stopped (16 weeks) (FIG. 9(a)). When looking atpercentage of the different categories of sleepers the combo treatmentincreased the number of patients describing their sleep as ‘good’ anddecreasing the number of subjects who had ‘fairly bad’ sleep and this ismaintained even after the feeding has stopped (FIG. 9(b)). We alsoanalysed a sub group of IBS patients who had reported that they had‘bad’ sleep at baseline, and this was reflected in a global score ofPSQI of greater than 5. Subjective sleep quality scores improved frombaseline to the end of the intervention (8 weeks) in this group (globalPSQI score of over 5) and maintained their effect after feeding hadstopped (16 weeks) (FIG. 10(a)). When looking at percentage of thedifferent categories of sleepers the combo treatment increased thenumber of patients describing their sleep as ‘fairly good’ anddecreasing the number of subjects who had ‘fairly bad’ sleep and this ismaintained even after the feeding has stopped (FIG. 10(b)).

In this IBS study 33 of the 40 patients had a global PSQI score of over5 and have so been characterised as bad sleepers which agrees with theliterature which shows that IBS patients have poor sleep quality. Aftercombo administration there was a significant improvement in sleepquality as measured by the PSQI in these IBS patients and this effect ismaintained 8 weeks after the combo administration.

The beneficial effect of combo product on sleep quality is indicative ofthe potential benefits of Bifidobacterium longum 1714™ strain forimproving sleep quality, especially in IBS patients.

The effect of 1714™ strain on sleep quality was assessed in healthyhuman subjects, and chronically stressed healthy subjects and IBSpatients and it has shown benefit in all three instances.

The invention is not limited to the embodiments hereinbefore described.

REFERENCES

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BUYSSE D J, REYNOLDS C F, MONK T H, BERMAN S R, KUPFER D J 1989 ThePittsburgh Sleep Quality Index: a new instrument for psychiatricpractice and research. Psychiatry Res. May; 28(2): 193-213.

BUYSSE D J, REYNOLDS C F 3RD, MONK T H, HOCH C C, YEAGER A L, KUPFER DJ. 1991 Quantification of subjective sleep quality in healthy elderlymen and women using the Pittsburgh Sleep Quality Index (PSQI) Sleep.August; 14(4):331-8.

DINAN T G, STANTON C, CRYAN J F. 2013 Psychobiotics: a novel class ofpsychotropic. Biol Psychiatry.; 74(10):720-6,

FILLINGIM R B, OHRBACH R, GREENSPAN J D, KNOTT C, DUBNER R, BAIR E,BARAIAN C, SLADE G D, MAIXNER W. 2011 Potential psychosocial riskfactors for chronic TMD: descriptive data and empirically identifieddomains from the OPPERA patient-control study. J Pain.; 12:T46-T60.

HERMAN C R, SCHIFFMAN E L, LOOK J O, RINDAL D B 2002 The effectivenessof adding pharmacologic treatment with clonazepam or cyclobenzaprine topatient education and self-care for the treatment of jaw pain uponawakening: a randomized clinical trial. J Orofac Pain.; 16:64-70.

INSTITUTE OF MEDICINE (US) COMMITTEE ON SLEEP MEDICINE AND RESEARCH;COLTEN HR, ALTEVOGT BM, EDITORS. 2006 Sleep Disorders and SleepDeprivation: An Unmet Public Health Problem. Washington (DC): NationalAcademies Press (US).

PINTO-SANCHEZ, M. I., HALL, G. B., GHAJAR, K., NARDELLI, A., BOLINO, C.,LAU, J. T., MARTIN, F. P., COMINETTI, O., WELSH, C., RIEDER, A.,TRAYNOR, J., GREGORY, C., DE PALMA, G., PIGRAU, M., FORD, A. C., MACRI,J., BERNER, B., BERGONZELLI, G., SURETTE, M. G., COLLINS, S. M.,MOAYYEDI, P. & BERCIK, P. 2017. Probiotic Bifidobacterium longum NCC3001Reduces Depression Scores and Alters Brain Activity: a Pilot Study inPatients With Irritable Bowel Syndrome. Gastroenterology.

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1-16. (canceled)
 17. A method of treating a human subject, the method comprising administering to the subject a formulation comprising Bifidobacterium longum strain NCIMB 41676 and an ingestible carrier, wherein the formulation comprises more than 10⁶ cfu of the Bifidobacterium longum strain, and wherein the formulation improves sleep quality in the subject as measured by a decrease in amount of time to fall asleep, an increase in sleep duration, a decrease in sleep disturbance, a decrease in daytime dysfunction due to sleep disturbance, or a combination thereof.
 18. The method of claim 17, wherein the formulation comprises 10⁷ to 10¹⁰ cfu of the Bifidobacterium longum strain.
 19. The method of claim 17, wherein the formulation comprises 10⁸ to 10⁹ cfu of the Bifidobacterium longum strain.
 20. The method of claim 17, wherein the formulation further comprises a prebiotic.
 21. The method of claim 17, wherein the formulation is in the form of a capsule, a tablet or a powder.
 22. The method of claim 17, wherein the formulation includes a controlled release coating.
 23. The method of claim 17, wherein the formulation is in the form of a food product.
 24. The method of claim 23, wherein the food product comprises an acidified milk, a yoghurt, a frozen food, a gum, a candy, a milk powder, a milk concentrate, a cheese spread, a nutritional composition, a nutritional supplement, a cereal bar, a dressing or a beverage.
 25. The method of claim 17, wherein the formulation further comprises a protein, a peptide, a vitamin, or a combination thereof.
 26. The method of claim 17, wherein the formulation further comprises a drug entity or a biological compound.
 27. The method of claim 17, wherein the subject is a baby.
 28. The method of claim 17, wherein the subject is an elderly person.
 29. The method of claim 17, wherein the subject has irritable bowel syndrome.
 30. The method of claim 17, wherein the formulation is administered to the subject once per day for at least one week.
 31. A method of treating a human subject, the method comprising administering to the subject a formulation comprising Bifidobacterium longum strain NCIMB 41676 and an ingestible carrier, wherein the formulation comprises more than 10⁶ cfu of the Bifidobacterium longum strain, wherein the Bifidobacterium longum strain is in powder form, and wherein the formulation improves sleep quality in the subject as measured by a decrease in amount of time to fall asleep, an increase in sleep duration, a decrease in sleep disturbance, a decrease in daytime dysfunction due to sleep disturbance, or a combination thereof.
 32. The method of claim 31, wherein the formulation is in the form of a capsule, a tablet, or a sachet.
 33. The method of claim 31, wherein the formulation comprises 10⁷ to 10¹⁰ cfu of the Bifidobacterium longum strain.
 34. The method of claim 31, wherein the subject is a baby.
 35. The method of claim 31, wherein the subject is an elderly person.
 36. A method of treating a human subject, the method comprising administering to the subject a formulation comprising Bifidobacterium longum strain NCIMB 41676, wherein the formulation improves sleep quality in the subject as measured by a decrease in amount of time to fall asleep, an increase in sleep duration, a decrease in sleep disturbance, a decrease in daytime dysfunction due to sleep disturbance, or a combination thereof. 